Design an nd Synthessis of novvel metal complex-p c protein coonjugatio on agentts for App plications in bio-im maging W Wang Tao o Supervisoor: A/P Tanja Weiil N NATION AL UNIV VERSITY Y OF SINGAPORE E 2010 Design and Synthesis of novel metal complex-protein conjugation agents for Applications in bio-imaging WANG TAO (BSc, Sichuan University2008 ) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements ACKNOWLEDGEMENTS I would like to take this opportunity to express my heartfelt gratitude to those who help me in research and writing thesis. A special acknowledgement is given to my supervisor, Associate Professor Tanja Weil, who gives me kind encouragement and useful instructions all through my research. She is willing to discuss the difficulties encountered in my project and always give creative suggestions. She is also very kind and considerable, making our group like a sweet and happy family. I would like to my sincerely thank my colleagues Dr. Kuan Seah Ling, Wu Yuzhou, Chen Xi, Goutam Pramanik, Ng Yuen Wah David, They offer me invaluable advice and appreciate help throughout the project. I would like to my parents and friends for their consideration and motivation. Last but not least, I would like to thank the chemistry department of NUS for giving me the opportunity to undertake this project. i Table of Contents TABLE OF CONTENTS Acknowledgements i Table of Contents ii List of Figures v List of Schemes vi Index of Abbreviations vii Abstract ix Chapter 1. Introduction 1 1.1 Metal Complex for MRI or PET 1 1.2 The Biological Function of Folic Acid 4 1.3 The Biological Significance of Somatostatin 6 1.4 Chemical Modification of Proteins 9 1.5 Site-specific Intercalation Into Protein Using a Three-carbon Bridge 13 1.5.1 Disulfide Bonds in Therapeutically Relevant Proteins 13 1.5.2 Reduction of Disulfides and Disulfide Site-specific Intercalation 14 1.6 Design of biocompatible metal-complex protein conjugate Chapter 2. Project Aim and Design 16 18 ii Table of Contents Chapter 3. Results and Discussion 20 3.1 Synthesis of 1, 4, 7-tris (tert-butoxycarbonylmethyl)-1, 4, 7, 10tetraazacyclododecane 3 (DO3tBu) (3) 20 3.2 Synthesis of tert-butyl 2, 2’, 2’’-(10-(2-oxo-2-(prop-2-ynylamino) ethyl) -1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetate (8) 22 3.3 Synthesis of tert-butyl 2,2'-(4-(2-tert-butoxyallyl)-10-(6-(2,5-dioxo-2,5dihydro-1H-pyrrol-1-yl)hexanoyl)-1,4,7,10-tetraazacyclododecane-1,7diyl)diacetate (10) 23 3.4 Preparation of DOTA-Folate Conjugate 24 3.5 Synthesis of Tailored Linker (16) 26 3.6 Synthesis of Water Soluble Intercalator (21) 27 3.7 Intercalation of Somatostatin 31 Chapter 4. Experimental 33 4.1 General Procedures 33 4.2 Synthesis of 1, 4, 7-tris (tert-butoxycarbonylmethyl)-1, 4, 7, 10tetraazacyclododecane (DO3tBu) (8) 4.3 Synthesis of 2-bromo-N-(prop-2-ynyl) acetamide (6) 34 36 4.4 Synthesis of Tert-butyl 2,2',2''-(10-(2-oxo-2-(prop-2-ynylamino)ethyl)1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (8) 4.5 Synthesis of 6-maleimideocaproic acid (9) iii 37 38 Table of Contents 4.6 Synthesis of Tert-butyl 2, 2’-(4-(2-tert-butoxyallyl) -10-(6-(2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoyl) -1, 4, 7, 10-tetraazacyclododecane-1, 7-diyl) diacetate (10) 40 4.7 Synthesis of Folate-NHS (11) 41 4.8 Synthesis of Folate-DOTA (13) 42 4.9 Synthesis of Folate-DO3tBu (12) 43 4.10 Synthesis of Mannich Salt (14) 46 4.11 Synthesis of Bis-disulfide (15) 47 4.12 Synthesis of Bis-sulfone (16) 48 4.13 Synthesis of Bromoethyl-bis-sulfide (18) 50 4.14 Synthesis of Piperazine-bis-sulfide (23) 52 4.15 Synthesis of Tert-butyl 4-(2-aminoethyl) piperazine-1-carboxylate (21) 53 4.16 Synthesis of Tert-butyl 4-(2-aminoethyl) piperazine-1-carboxylate (31) 54 Chapter 5. Conclusion 56 Chapter 6. References 59 Appendix 68 iv LIST OF FIGURES Figure 1.1 Page The structure of 1, 4, 7, 10-tetraazacyclododecane -1, 4, 7, 10-tetraacetic acid (DOTA) and folic acid 2 1.2 FR-mediated endocytosis of a folic acid conjugate 5 1.3 [18F]-FDG microPET (middle), MR (right), and [66Ga]Ga-DF-Folate microPET (left) images of mice with subcutaneous folate-receptor-positive human KB cell tumor xenografts in their intrascapular region 6 1.4 The structure of somatostatin 7 1.5 (a) Site-specific modification of protein yields homogenity and (b) Non-specifcification modification of protein results heterogeneity  9 1.6 The bifuncional molecule consisting DOTA complex and folic acid 18 1.7 The bifunctional molecule consisting DOTA complex and folic acid 19 1.8 The bifuncional molecule with maleimide-DOTA complex and 1.9 Protein with free thiol group e.g. BSA 19 The side product in the synthesis of compound 18 29   v    LIST OF SCHEMES Scheme 2.1 Page Non-specific modification of a) lysines and b) cysteines residue on proteins 2.2 11 Mechanism for conjugating a three-carbon bridge to a native disulfide bond. 2.3 15 Synthesis of tri-tert-butyl 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (DO3tBu) 2.4 20 Synthesis of tert-butyl 2, 2', 2''-(10-(2-oxo-2-(prop-2-ynylamino) ethyl) -1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetate (8) 22 2.5 Synthesis of DOTA-maleimide deriveritives (10) 23 2.6 The first procedure to synthesize Foate-DOTA (13) conjugates 24 2.7 The second procedure to synthesize Foate-DOTA conjugates 25 2.8 Synthesis of bis-disulfone (16) used to combine metal complex and biomelocule 26 2.9 The second method to synthesized bis-disulfone (16) 27 2.10 Synthesis of water soluble intercalator piperazine bis-sulfone (21) 28 2.11 The second synthetic route towards the water soluble intercalator (21) 29 2.12 The third synthetic route of water soluble intercalator (21) 30 2.13 Intercalation of Somatostatin 31 2.14 Proposed synthetic route for somatostatin DOTA conjugate 58 vi    INDEX OF ABBREVIATIONS CT Computed Tomography d doublet DCC N,N'-dicyclohexylcarbodiimide DCM Dichloromethane (Methylene Chloride) DIEA N,N-Diisopropylethylamine DMAP 4-Dimethylaminopyridine DMF Dimethylformamide DMSO Dimethylsulfoxide DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid EA Ethyl Acetate EDC 1-ethyl-3-3(3-dimethylaminopropyl) carbodiimide hydrochloride ESI Electron Spray Ionization Fab Fast Atom Bombardment FR Folate Receptor HBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium Hexafluorophosphate IT-TOF-MS Ion trap & Time-of-flight Mass Spectrometry LC-MS Liquid Chromatography & Mass spectrometry MIBK  4-Methyl-2-pentanone MRI Magnetic Resonance Imaging PET Positron Emission Tomography vii   q quartet s singlet SPECT Single-photon-emission Computed Tomography t triplet t-Bu tertiary butyl THF Tetrahydrofuran TLC Thin-Layer Chromatography UV Ultraviolet viii   Abstract Abstract Targeting particular cells or tissues for imaging e.g. proliferative cells or for transporting drug molecules plays a vital role in cancer treatment and represents an area of high scientific interest. In order to contribute to a better detection of proliferative cells, a sophisticated metal-DOTA imaging agent was designed that is able to specifically interact with disulfide bridges of proteins by intercalating into accessible disulfide bridges via two sequential Michael addition-elimination reactions. Such DOTA-protein conjugates are highly versatile since they can be labeled with 68 Ga for PET or paramagnetic metals such as Gd(Ⅲ) for MR imaging. In addition, a folate-metal-DOTA conjugate has been prepared as well as a novel approach that facilitates somatostatin-metal-DOTA conjugates has been designed for targeted delivery and first attempts have been undertaken to achieve this challenging goal. Folic acid or somatostatin-DOTA conjugates require conjugation via a tailored linker. The synthesis of this linker moiety, the functionalization of the metal-DOTA complex and the conjugation approach is thoroughly investigated. Based on this strategy, sitedirected labeling of peptides or even larger proteins with a single accessible disulfide bond such as antibodies becomes feasible. Future work will focus on the in vitro or in ix Abstract vivo evaluation of the Folic acid-DOTA derivative. In addition, a larger number of gadolinium complexes may be attached to proteins with multiple disulfide bridges which may yield improved contrast and low detection limit. x Chapter 1 Chapter 1. 1.1. Introduction Introduction Metal Complex for MRI or PET Molecular imaging is one of the most exciting and rapidly growing areas of science as it enables the characterization and quantification of biological processes at the cellular and subcellular level in living subjects in an intact manner[1]. It utilizes specific molecular probes as well as intrinsic tissue characteristics as the source of image contrast, and offers the opportunity for an improved understanding of integrative biology, earlier detection and characterization of diseases, and facilitates a better evaluation of therapeutic treatment[2]. The imaging modalities can be broadly divided into two categories: anatomical and molecular techniques. Examples of anatomical imaging technologies include computed tomography (CT) and magnetic resonance imaging (MRI), which are characterized by high spatial and temporal resolutions. On the other hand, molecular techniques such as positron-emission tomography (PET) and single-photonemission computed tomography (SPECT) offer excellent sensitivity and often provide important biochemical information on pathological conditions [3, 4]. 1 Chapter 1 Introduction Figure 1. The structure of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and folic acid In molecular imaging techniques, the contrast media play an important part in improving the sensitivity, resolution of images and target specificity at the molecular/cellular level. However, the toxicity of the contrast agent is a major concern in its application. For example, lanthanide ions are widely used as MRI contrast agents[5], radioactive tracers[6], and optical imaging probes[7, 8]. Nonetheless, free lanthanide ions often exhibit high toxicity in vivo. To circumvent this problem, chelating agents are extensively used to coordinate to these metal ions and thus minimize their toxicity. Among various chelating agents, macrocyclic 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid (DOTA) (Figure 1) is one of the widely used ligand in molecular imaging as it outperforms other agents in the ability to form complexes with a large number of transition and lanthanide metal ions with high thermodynamic stability and kinetic 2 Chapter 1 Introduction inertness[9]. DOTA complexes, depending on the metal ions, are mainly used in three areas: magnetic resonance imaging [Gd(Ⅲ), Eu(Ⅲ)], nuclear imaging[111In(Ⅲ), 68Ga(Ⅲ), 64/67 Cu(Ⅱ)], and therapeutic radiopharmaceuticals [90Y(Ⅲ), 177 Lu(Ⅲ)]. Low–molecular- weight contrast agents for MRI, such as the commercial agent Gd3+-DOTA (DotaremTM) display low relaxivity and extremely fast excretion rates in vivo. To improve the relaxivity, Gd(Ⅲ) chelates are conjugated to macromolecules, like proteins[10], micellar aggregates[11], dendrimers[12], or liposomes[13], thus extending the rotational correlation lifetime. But most of them are not able to differentiate between “healthy” and e.g. tumor cells thus preventing cell or tissue-specific molecular imaging. Nonetheless, remarkable progress has been made in recent years in the development of targeted contrast agents for diagnostic imaging that allows better differentiation [14, 15]. Various targeted contrast agents for MRI and PET have been reported which were synthesized via the conjugation of metal chelates to various biomolecules, including peptides[16], proteins[17], antibodies[18], oligonucleotides[19] and biotin/avidin[20]. These biomolecules are used as molecular imaging probes which show high binding affinity to the target receptors, antigens, and nucleic acids being specifically overexpressed in or on the targeted cells tissues. 3 Chapter 1 1.2. Introduction The Biological Function of Folic Acid Folic acid(Figure 1) is a water-soluble vitamin of the B-complex group and plays essential roles in numerous bodily functions by participating in the biosynthesis of nucleic and amino acids [21]. More importantly, it can be utilized for targeted delivery. Targeted delivery via selective cellular marker improves the efficacy and safety of the therapeutic and imaging agents. Among cellular surface targets, folate receptors (FR)-α is most promising and well-investigated in epithelial cancers. The other form of FR (FR-β) is present in myeloid leukemia and activated macrophages, increasingly recognized as a cellular target[22]. A variety of molecules including radioimaging agents, magnetic resonance imaging (MRI) contrast agents, chemotherapeutic agents, oligonucleotides, proteins, enzyme constructs for prodrug therapy, haptens, liposomes, nanoparticles and gene therapy vectors have been conjugated to folate for FR-targeted delivery[22]. FR is significantly upregulated in cancer cells and occurs at very low levels 4 Chaapter 1 Introoduction in most m normaal tissues [223-25]. Follate conjugaates bind FR F with higgh affinity and are inteernalized intto tumors viia receptor-m mediated enndocytosis (Figure ( 2). e of a folic acid a conjugaate[26] Fiigure 2. FR--mediated endocytosis The keyy part of tuumor-specifiic imaging is the speciificity of the targeting unit for mallignant cellls and the capacity of o the tum mor-specificc receptor to bind suufficient quanntities of the t imagingg agent to achieve high contrastt. In the caase of highh tumor speccificity , vvery low concentratio c on of the PET imagging agent is needed d; while micromolar cooncentration n is still reequired in MRI[27]. Generally, targeting imaging i nts should: (a) show high h affinitty (Kd100-fold over normal cells is preferreed), (c) reveeal rapid cleearance from m normal (rreceptor neggative) tissuues, and (d) be uptakenn in sufficcient quantiities by recceptor-exprressing tissuues to alloow high 5 Chapter 1 Introduction contrast[28]. The folic acid and receptor pair fulfill most of these requirements; hence they are an attractive ligand/receptor combination for targeted imaging. Folate-targeted conjugates of radionuclides (Figure 3), like 99mTc[29-31], 111In[32, 33], 66/67/68 Ga[34, 35] and 18 F[36], for SPECT and PET imaging have been developed and evaluated in preclinical and clinical studies. In addition, a few folate-targeted MRI contrast agents have also been reported [37, 38]. Figure 3. [18F]-FDG microPET (middle), MR (right), and [66Ga]Ga-DF-Folate microPET (left) images of mice with subcutaneous folate-receptor-positive human KB cell tumor xenografts in their intrascapular region[39]. 1.3 The Biological Significance of Somatostatin Somatostatin(Figure 4) is a cyclic tetradecapeptide hormone. It is found in multiple sites throughout the nervous system, including the cerebral cortex, the brain 6 Chaapter 1 Introoduction stem m, the gastrrointestinal tract, and the t pancreaas. It plays many diverse roles inncluding inhiibition of enndocrine annd exocrine secretions, modulationn of neurotrransmission n, motor and cognitive ffunctions, innhibition off intestinal m motility, absorption off nutrients annd ions, vasccular contraactility, and cell prolifeeration[40]. F Figure 4. Thhe structuree of somatosstatin Somatoostatin mediiates its bioological effeects throughh interactioon with a faamily of s n receptors expressed by a varieety of norm mal and maalignant fivee specific somatostatin tissuues[40]. Som matostatin receptors r haave been iddentified by classical biiochemical binding 7 Chapter 1 Introduction techniques and in vitro autoradiography on a variety of human tumors, such as pituitary tumors, endocrine pancreatic tumors, carcinoids, paragangliomas, meningiomas, brain tumors (astrocytomas), neuroblastomas, and some human breast cancers[41]. Therefore, somatostatin/somatostatin receptor system is studied intensively in contrast-enhanced diagnostic imaging and targeted therapy of tumors. The exact mechanism of somatostatin antineoplastic activity is unknown, but some possibilities are: (1) a direct antiproliferative effect by blockade of mitogenic growth signal or induction of apoptosis through interaction with somatostatin receptors; (2) inhibition of secretion of gastrointestinal hormones thought to be important in tumor growth; and (3) reduction or inhibition of secretion of growth-promoting hormones and growth factors which stimulate the growth of cancers[42]. A major progress is made by introducing radiolabelled somatostatin for diagnosis and treatment of cancers. Somatostatin acts as a bullet to specifically target a maligant tissue with high affinity through interaction with somatostatin receptors. 8 Chapter 1 1.4. Introduction Chemical Modification of Proteins There is an increasing interest in protein conjugates for diagnosis and therapy. Even though proteins often display limited pharmacokinetics, low proteolytic stabilities and the possibility to elicit immune responses, there have been successful attempts of converting proteins into efficient drug delivery systems or imaging agent [43]. Their low nanometer sizes, highly defined structures, biodegradability and the presence of a high number of functional groups available for chemical modifications make them attractive for applications in targeted drug delivery and bioimaging. Figure 5. (a) Site-specific modification of protein yields homogenity and (b) nonspecifcification modification of protein results heterogeneity[44] 9 Chapter 1 Introduction Protein conjugates can be prepared via chemical modification and bioengineering techniques. Chemical modification approaches can be divided into two major categories; site-specific and non-specific protein functionalization(Figure 5). Classical non-specific protein conjugation techniques typically involve electrophilic reagents targeting the nucleophilic functional groups of lysine (Scheme 1), cysteine, aspartic acid or glutamic acid side chains, generally providing a heterogeneous mixture of proteins modified to a different extent and at variable locations in the protein conjugates[44, 45]. A more specific strategy represents the modification of cysteine residues (Scheme 1) through alkylation with iodoacetamide reagents, disulfide exchange and Michael addition with maleimides[46-48]. Since free cysteine groups are rare and often inaccessible, they can be engineered into the protein as point mutations using molecular biological techniques. Such approaches are usually demanding and expensive and point mutation may have a negative impact on protein function by altering its structure. Moreover, introducing an accessible free thiol group often leads to disulfide scrambling, protein misfolding and an increased tendency to form aggregates during purification. Still, 10 Chapter 1 Introduction thiol-specific modifications play an important role due to the potential for high-yield reactions (e.g. Michael reactions), as well as the propensity for addressing cysteine groups selectively without targeting other amino acids. Scheme 1. Non-specific modification of a) lysines and b) cysteines residue on proteins In recent years, significant progress has been made to develop improved strategies for selective and efficient protein chemistry and thus more well-defined protein conjugates[47, 48]. New means have been established for the modification of tyrosine and tryptophan, usually by applying transition-metal-mediated processes that are 11 Chapter 1 Introduction compatible with aqueous conditions[45]. Tyrosine residues are modified via a threecomponent Mannich reaction with aldehydes and anilines[46, 49]. Targeting tryptophan residues has been developed by employing rhodium carbenoids in acidic condition (pH≈ 2), which may affect the structure of some protein[50]. As hydrophobic amino acids are generally buried within the protein scaffold, controlled single-site modification of tyrosines and tryptophans is possible in some cases by improving surface accessibility often via point mutations[51]. Probably the most elaborate method to site-specifically modify proteins involves the introduction of non-canonical amino acids (rNCAA) into proteins[45]. Here, by chemically attaching the desired rNCAA to suppressor tRNA and then placing the amber codon at the desired position in the mRNA, a number of rNCAA have been incorporated at different positions into the protein sequence. Successful examples of rNCAA that are incorporated into the protein sequence include p-iodotyrosine, which undergoes Pdcatalyzed alkenylation ( Mizoroki-Heck reaction ) or alkynylation ( Sonogashira reaction ) reactions from the protein surface[52], Stille coupling using organotin derivatives as well as Suzuki reactions utilizing boronic acids and esters[45]. Previously, rNCAA with an azido or ethynyl group has been incorporated into different proteins[53]. Azide-alkyne 12 Chapter 1 Introduction [3+2] cycloaddition are conducted in the presence of Cu (I) as catalyst, yielding exclusively the 1,4-substituted triazole isomer. These reactions proceed rapidly in water, and provide excellent chemoselectivity and regioselectivity [53]. 1.5. Site-specific Intercalation into Proteins using a Three-Carbon Bridge 1.5.1 Disulfide Bonds in Therapeutically Relevant Proteins In general, free and accessible cysteine residues are rare[54] and liable to pair up to form disulfides bridges [55, 56]. Disulfide bonds influence the physio-chemical and biological properties of proteins in many subtle and complex ways[57]. They are either buried within the protein’s folding region or on its solvent accessible surface[58]. Solvent-accessible disulfides can be selectively approached by tailored reagents and can be chemically modified. As accessible disulfides primarily contribute to the stability of a protein rather than to its structure or biological function[59] it is feasible to intercalate into this bond by tailored reagents without a loss of either structure or function. Previously, protein databases and molecular modeling programs have been used to 13 Chaapter 1 Introoduction estim mate the reelative surfface accessiibility of disulfide d bonds and prredict the potential p influ uence of innserting a 3-carbon bridge. b In pparticular, the t group of o Brocchin ni et al. evalluated proteein databasses and conncluded thaat most theerapeuticallyy relevant proteins p have at least one o disulfidde bond close to the surface thaat can be ttarget for in nserting mical modifications[600]. chem 1.5..2 Reducttion of Disu ulfides and Disulfide Site-specific S c Intercalaation heme 2. Mecchanism forr conjugatinng a three-caarbon bridgge to a nativve disulfide bond. b Sch Selectivve reductionn of accesssible disulfiide bonds is i tolerated by many proteins p undder mild connditions wiithout the use u of denaaturants. Diithiothreitoll (DTT) or tris (2carbboxyethyl) phosphine hydrochlooride (TCE EP.HCl) iss typicallyy used to reduce acceessible disuulfide bondss simultaneo ously maintaaining the protein’s p terrtiary structuure [60]. 14 Chapter 1 Introduction Consequently, the two free cysteine sulfur atoms are available for chemical modification for instance, via Michael addition reactions. After the mild reduction of the accessible disulfide in a native protein, the two free thiol groups are ready for the site-specific intercalation (in case the protein offers just a single accessible disulfide bridge). Generally, the intercalating reagents comprise of an electron-withdrawing group (e.g., carbonyl group), an α,β-unsaturated double bond and a α , β ’sulfonyl group that is susceptible to elimination as sulfinic acid[61]. The conjugated double bond in the mono-sulfone allows for a sequence of interactive and sequential addition-elimination reactions[61]. (Scheme 2) The site-specific intercalation into a disulfide bond differs from other thiolspecific modifications. For the commonly used methods, each sulfur react separately and independently with di-thiol reagents; while the two cysteines of the original disulfide bond are covalently reconnected through a three-carbon methylene bridge. Since other reagents used to modify disulfides like two maleimides or two vinyl sulfones are chemically independent, they cannot undergo controlled bis-alkylation by sequential addition-elimination reactions[60]. In addition, this method of using a intercalating agent is highly attractive since it is crucial to preserve a protein’s tertiary structure after a mild 15 Chapter 1 Introduction reduction of an accessible disulfide to (i) allow the free thiol groups to be spatially close to each other, (ii) minimize any chance of the irreversible denaturation and aggregation of the protein, and (iii) prevent disulfide scrambling reactions if more than one disulfide bond is reduced[61]. 1.6. Design of biocompatible metal-complex protein conjugates In order to design biocompatible metal-complex protein conjugates which can achieveprecise intracellular delivery and improved sensitivity for imaging, two components are required, namely, an imaging agent and an efficient targeting entity. There are several strategies that can be adopted. One of which is the monofunctionalization of DOTA, followed by the combination of an imaging group and a cell targeting moiety via amide bond formation. The other method is the synthesis of a tailored linker which can intercalate into the cell targeting entity. Then the two parts can be combined via click reaction or Michael addition. As a targeting unit, folic acid interacts with folate receptors that are overexpressed in certain cancer cell lines. Similarly, somatostatin interacts with G-protein-coupled somatostatin receptors. Metal-DOTA and somatostatin could be conjugated via a tailored linker as outlined in the second method. DOTA-Folate (Scheme 7, 8) and DOTA-somatostatin (Scheme 14) conjugates will be prepared, and their biological properties will be investigated via in vitro experiments. 16 Chapter 1 Introduction When 68Ga labeled conjugates have been synthesised, the tracers can be studied via PET using small animal models, such as rats, implanted with tumor cells. The PET biodistribution data can be obtained and compared with other imaging modes. Similarly, MR imaging can also be achieved by labeling the conjugates with paramagnetic metals Gd(Ⅲ). Based on this strategy, labeling of larger proteins bearing several disulfide bonds will be explored in order to attach a larger number of gadolinium or gallium complexes which might improve the contrast and detection limit. 17 Chapter 2 Chapter 2. Project Aim and Design Project Aim and Design (Ⅲ) (Ⅲ) Figure 6. The bifuncional molecule consisting DOTA complex and folic acid The aim of my project is to synthesize novel bifunctional molecules that allow imaging via a DOTA unit connected to a cell targeting folic acid group. The bifunctional molecule consisting of the DOTA complex and folic acid moiety is shown in Figure 6. Considering that folic acid is only limited to certain cancer cell lines and that generally, proteins such as antibodies and peptides such as somatostatin also represent attractive entities allowing targeted delivery. The final structure of the bifunctional molecule consisting DOTA complex and somatostatin is shown in Figure 7. However, the defined and ideal site-directed functionalization is a key concern. Therefore, the disulfide site specific intercalation is proposed to allow the effective attachment of a DOTA group. In 18 Chaapter 2 Prooject Aim and d Design addition, the deesign of com mpletely noovel intercaalation agennts with impproved solubbility to faciilitate waterr-chemistry will be expplored. Figure 7. The bifuncctional moleecule consissting DOTA A complex aand folic accid In the ccase where the t protein or o peptide has h an accesssible cysteiine residue, such as bovvine serum albumin (BSA), sitte-directed functionaliization cann be achieeved by conjjugating wiith maleimidde-DOTA complex c (Fiigure 8). O O N O N O BSA N S O N O N O O O = o Gd (Ⅲ) or 68 Ga (Ⅲ) Fiigure 8. The bifuncion nal moleculee with maleiimide-DOT TA complexx and Protein n with free thiol t group e.g. BSA 19 Chapter 3 Results and discussion Chapter 3. 3.1 Results and discussion Synthesis of 1, 4, 7-tris (tert-butoxycarbonylmethyl)-1, 4, 7, 10- tetraazacyclododecane (DO3tBu) (3) O O H N t-BuO NH NH O Br N 2 NH trithylamine, chloroform, 64% NH O O 3 O O H N Side Product: O N 1 O N N NH O N 4 N O O O O O N O N O N 5 O O Scheme 3. Synthesis of tri-tert-butyl 2,2',2''-(1,4,7,10-tetraazacyclododecane-1,4,7triyl)triacetate (DO3tBu) To efficiently attach the DOTA chelator to biomolecules, it should be functionalized with a reactive group that allowed covalent bond formation to the biomolecules. Since amine groups are abundant in biomolecules, and the conversion of a pendant carboxylic acid of DOTA into a carboxamide minimally affected the stability of 20 Chapter 3 Results and discussion the conjugating metal complex, a straightforward approach would be to the conjugation of DOTA derivatives to biomolecules through a physiologically stable amide bond[40]. In this context, the synthesis of DOTA-monoamide derivatives by an efficient strategy was the main focus. The synthesis of 1, 4, 7-tris (tert-butoxycarbonylmethyl)-1, 4, 7, 10tetraazacyclododecane (DO3tBu) (3) was described in this section. In the initial trials, chloroform and triethylamine were used as the solvent and base respectively (Scheme 3). According to literature, triethylamine exhibited greater steric hindrance and would lead to tri-substituted cyclen as a main product [62]. On the contrary, analysis with the LC-IT-TOF-MS spectra (Appendix 29) indicated that the reaction mixture contains comparable quantity of 3, 4 and 5, indicating that the selected reaction condition was not optimal for achieving selectivity. Furthermore, the side product 5 could not be separated from the target molecule, 3, even after column chromatography, as indicted in the ESI-MS spectrum. Hence, a different protocol was attempted by using acetonitrile as solvent and sodium bicarbonate as base (Scheme 4). The reaction mixture was monitored using LCIT-TOF-MS (Appendix 29). The spectrum obtained indicated that this approach was also lacking in terms of selectivity, and significant amount of by-products 4 and 5 were 21 Chapter 3 Results and discussion formed. The pure compound 3 was obtained by column chromatography using CDCl3/MeOH= 10:1, while the use of DCM/MeOH=10:1 couldn’t afford the desired separation. 3.2 Synthesis of Tert-butyl 2, 2’, 2’’-(10-(2-oxo-2-(prop-2-ynylamino) ethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetate (8) Scheme 5 Synthesis of tert-butyl 2,2',2''-(10-(2-oxo-2-(prop-2-ynylamino)ethyl)- 1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (8) An alkyne-functionalized DOTA derivative 8 (Scheme 5) was prepared since it could specifically label azido-modified biomolecules by Cu( Ⅰ ) catalyzed [3+2] cycloaddition (click chemistry). The purification of compound 8 was non-trivial, as the spot of compound 8 could not be resolved from the side product on the TLC plate. The 22 Chapter 3 Results and discussion spot of compound 8 could be confirmed by staining with KMnO4 or I2, whereas the side product could only be stained by I2. Therefore, the yield of 8 from this reaction was low. Both the 1H NMR spectrum and 13 C NMR spectra of compound 8 were in agreement with the literature [63]. 3.3 Synthesis of tert-butyl 2, 2’-(4-(2-tert-butoxyallyl)-10-(6-(2, 5-dioxo-2, 5dihydro-1H-pyrrol-1-yl) hexanoyl)-1, 4, 7, 10-tetraazacyclododecane-1, 7-diyl) diacetate (10) O O O O O + H2N O O OH OH N OH 65% O O 9 O O N N NH O N O O O O EDC, DMAP,DCM, 40% N O N O N N O 10 O O N O O Scheme 6. Synthesis of DOTA-maleimide deriveritives (10) In some cases, site-specific labeling of cysteine resides of proteins via Michael addition was feasible, and therefore, an additional maleimide functionalized DOTA 23 Chapter 3 Results and discussion derivative 10 was prepared (Scheme 6). When EDC and DMAP were added to compound 9 dissolved in DCM, the color of the solution changed from colorless to pink. This indicated the possibility of a side reaction which formed a colored impurity. 3.4 Preparation of DOTA-Folate Conjugate Scheme 7. The first route to synthesize Folate-DOTA (13) conjugate 24 Chapter 3 Results and discussion Scheme 8. The second route to synthesize Folate-DOTA conjugate Schemes 7 and 8 elaborated two facile ways to synthesize folate-DOTA conjugates and the biological evaluation of these bifunctional molecules are currently in progress. All the reactions were monitored using LC-IT-TOF-MS. The molecular mass of folic acid and Folate-NHS (11) corresponded to 441 g/mol and 538 g/mol. The LC profile indicated that the Folate-NHS derivative 11 was formed since a signal at 538 g/mol was observed. The major component detected corresponds to folic acid. However, since 25 Chapter 3 Results and discussion folate-NHS could be easily hydrolyzed in the LC conditions, it is highly plausible that the content of Folate-NHS (11) was under-estimated. For further reactions, excess of the Folate-NHS containing some folic acid was used. The main problem with the folate derivatives was their poor solubility in most organic solvents. They were soluble in DMSO and sparingly soluble in DMF; therefore conventional normal phase column chromatography was ineffective. Preparative reversed phase HPLC was used instead to isolate the products. 3.5 Synthesis of Tailored Linker (16) Scheme 9. Synthesis of bis-disulfone (16) 26 Chapter 3 Results and discussion The tailored linker, 16, could specifically intercalate into disulfide bridges of proteins via two consecutive Michael addition reactions and the synthesis is depicted in Scheme 9. The linker could also be modified with additional functionality to allow for a covalent linkage to metal chelates. The oxidation from bis-disulfide 15 to bis-disulfone could also be achieved by using H5IO6 and CrO3 (Scheme 10) and both methods of oxidation were attempted. Scheme 10. Alternative method to convert bis-disulfide to bis-disulfone (16) The reaction of 15 with H5IO6 and CrO3 was monitored by cospotting with bisdisulfone and bis-disulfide on TLC (Hexane/EA=1:1). On the TLC, the reaction mixture had three spots which correlate in terms of retention factor to a mixture of the starting material 15, reaction intermediate 17 and the product 16 indicating that the oxidation was incomplete. In comparison, oxidation utilizing oxone does not form the intermediate 17 and thus this presents a more convenient and efficient way to bring the reaction to completion. 27 Chapter 3 3.6 Results and discussion Synthesis of Water Soluble Intercalator (21) Since the intercalation to biomolecules has to be in aqueous condition and the water solubility of the mono-sulfone affected the rate and efficiency of the intercalation, intercalators with improved water solubility had been designed. Here, bis-disulfide 15 was modified and a piperazine group was introduced which could be protonated in order to achieve improved water solubility (Scheme 11). The modification was performed on the compound 15 since the bis-disulfide is more stable than the bis-disulfone, 16. Scheme 11. Synthesis of water soluble intercalator piperazine bis-sulfone (21) 28 Chapter 3 Results and discussion In the synthesis of compound 18, the ESI-MS spectrum showed a major mass peak of 462 which suggests that the alkyl bromide was labile under the reaction condition and had dissociated to form compound 22 (Figure 9). The FAB-MS spectrum displayed two peaks, 462 and 565 which proves that the alkyl bromide is labile. Figure 9: The side product in the synthesis of compound 18 The reaction route was modified as shown below (Scheme 12). Bis-disulfide 15 was reacted directly with 2-piperazinoethylamine under standard HBTU coupling conditions. Thereafter, the resulting compound 23 could be oxidized to bis-disulfone 21 using oxone. 29 Chapter 3 Results and discussion Scheme 12. The second synthetic route towards the water soluble intercalator (21) When bis-disulfide 15 was converted to compound 23, the product was monitored by TLC (Hexane/EA =1:1).The major spot corresponded to a retention factor of 0.8 which was unlikely to be the desired product. Upon analysing the reaction mixture using LC-IT-TOF-MS (Appendix 11), it was found that the desired piperazine bis-sulfide 23 was formed as the minor product and in addition, the peaks were not well resolved under several eluting conditions. The low yield of the reaction could be attributed to the chemoselectivity of the coupling reaction towards the free primary and secondary amine groups. Therefore, the reaction was modified to carry with the additional protection of the 2-piperazinoethylamine as shown below (Scheme 13). 30 Chapter 3 Results and discussion Scheme 13. The third synthetic route of water soluble intercalator (21) In the attempt to synthesize compound 26, several different conditions were attempted but the selectivity of the protection was not achieved. 3.7 Intercalation of Somatostatin Somatostatin was found to interact with G-protein-coupled somatostatin receptors which were abundant in various tumours, notably neuroendocrine tumours of which cacinoid tumour and phaeochromocytoma were encountered most in the clinical practice. 31 Chapter 3 Results and discussion The bifunctional linker, 15, was designed to conjugate to both the metal DOTA complex and somatostatin(Scheme 14). In this project, the linker 15 was successfully conjugated to DOTA giving compound 31, which should be subsequently converted to the bisdisulfone for intercalation into the somatostatin. However, compound 31 was unstable in the oxidation condition and degraded during the reaction. Scheme 14. Intercalation of Somatostatin 32 Chapter 4 Experimental Chapter 4. Experimental 4.1 General Procedures Unless otherwise noted, all operations were performed without taking precautions to exclude air and moisture. All solvents and reagents were purchased from commercial sources and were used without further purification. Reaction progress was monitored by thin layer chromatography (TLC) using Merck 60 F254 pre-coated silica gel plates illuminating under UV 254nm or using appropriate stains. Flash column chromatography was carried out using Merck silica gel 70-230 mesh. NMR spectra were measured on a Bruker ACF 300 and AMX 500 spectrometers and the shifts were referenced to residual solvent shifts in the respective deutero solvents. Chemical shifts are reported as parts per million referenced with respect the residual solvent peak. Mass spectra were acquired on a Finnigan Mat 95XL-T or a Finnigan Mat LCQ (ESI) spectrometer.LC-MS analysis was carried out using Shimadzu IT-TOF. 33 Chapter 4 4.2 Experimental Synthesis of 1, 4, 7-tris (tert-butoxycarbonylmethyl)-1, 4, 7, 10- tetraazacyclododecane (DO3tBu) (3) O O H N t-BuO NH NH O Br N NH trithylamine, chloroform, 64% NH O N 1 O O 3 O O H N Side Product: O N 2 N NH O N 4 N O O O O O N O N O N O O 5 Procedure 1 [64] Tert-butylbromoacetate (198 mg, 1.016 mmol, 3.5equiv) dissolved in 3mL of anhydrous chloroform was added dropwise to a mixture of 1,4,7,10- tetraazacyclododecane (cyclen) (50.0 mg, 0.2902 mmol, 1equiv) and triethylamine (294 mg, 2.902 mmol, 10equiv) in 5 mL of anhydrous chloroform under argon atmosphere. The reaction mixture was stirred for a further 20 h. The resulting solution was washed by water, and the organic phase was dried by Na2SO4. The solvent was removed, and the crude products was purified by column chromatography (DCM/MeOH=10:1). After 34 Chapter 4 Experimental purification, the product was still a mixture, containing the desired product (3) and tetratert-butyl 2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetate (5). ESI-MS (MeOH, 250oC): (+) m/z = 515[3+H], 651[5+Na]. Procedure 2: [65] Under argon atmosphere, cyclen (210 mg, 1.3 mmol, 1equiv) and sodium bicarbonate (350 mg, 4.2 mmol, 3.23equiv) were mixed in 150ml acetonitrile. Then, tertbutyl bromoacetate (820 mg, 4.2 mmol, 3.23equiv) in 50ml acetonitrile was added dropwise at RT for a period of 30 min. The reaction mixture was refluxed for 24 h at 87 ℃. The resulting precipitate was filtered and the solvent was evaporated. The crude product was first purified by recrystallization from toluene. A white powder was obtained and purified by column chromatography using chloroform/MeOH (v/v=10:1) as eluting solvent. 1, 4, 7-tris (tert-butoxycarbonylmethyl)-1, 4, 7, 10-tetraazacyclododecane (DO3tBu) (3) was isolated in 64% yield. 35 Chapter 4 1 Experimental H NMR (500MHz, CDCl3): δ 3.2402 (s, 4H), 3.1494 (s, 2H), 2.9540 (br s, 4H, ring- CH2), 2.7825 (br, 8H, ring-CH2), 2.7271 (br, 4H, ring-CH2), 1.3088 (s, 27H, Boc); (Appendix 14) 13 C NMR (500MHz, CDCl3): δ 28.19, 28.24, 47.51, 49.26, 51.40, 58.23, 81.69, 81.84, 169.63, 170.51; (Appendix 15) ESI-MS (MeOH, 250oC): (+) m/z= 515 [M+H]; (Appendix 16) 4.3 Synthesis of 2-bromo-N-(prop-2-ynyl) acetamide (6) Procedure [63] Aqueous 0.1M NaOH (4 ml) was poured on top of a solution of propargylamine (0.4 ml, 5.83 mmol) in 20ml DCM. Then, bromoacetyl chloride (6 ml, 18.7 mmol) was added by a syringe into the DCM layer, causing the immediate formation of a brown precipitate. The brown mixture was stirred vigorously for 2.5 h at RT during which time the precipitate re-dissolved. After the reaction was finished, the reaction solution was transferred to a separator funnel with excess DCM and water. The aqueous layer was 36 Chapter 4 Experimental isolated and extracted with DCM (5*10 ml). All organic fractions were combined and washed with 50 mM sodium carbonate (3*10 ml). The solution was dried by anhydrous magnesium sulfate and the solvent was removed under vacuum to form brown solid with 26 % yield. 1 H NMR (500MHz, CDCl3): δ 6.6396(s, 1H, NH), 4.0925(q, 2H, 2.67MHz, CH2NH), 3.8992(s, 2H, CH2Br), 2.2803(t, 1H, 2.64MHz, triple bond hydrogen); (Appendix 17) ESI-MS (MeOH, 250oC): (-) m/z=175[M-H]; (Appendix 18) 4.4 Synthesis of Tert-butyl 2,2',2''-(10-(2-oxo-2-(prop-2-ynylamino)ethyl)-1,4,7,10tetraazacyclododecane-1,4,7-triyl)triacetate (8) Procedure [63] A solution of DO3tBu 3 (59 mg, 0.1146 mmol), N-(2-propynyl)bromoacetamide (20 mg, 0.1146 mmol) and K2CO3 (16 mg, 0.1146 mmol) in DMF (1.2 ml) was stirred under argon at r.t. for 2 h. After removal of the solvent in vacuo, the residual mixture was 37 Chapter 4 Experimental purified by silica gel column using chloroform/methanol = 95 ： 5 → 9 ： 1. The compound 8 was isolated in 47% as brown solid. 13 C NMR (500MHz, CDCl3): δ172.30，171.93， 81.87， 80.86， 69.55， 56.10， 56.01， 55.70， 55.61，28.51，27.97，27.89; (Appendix 19) ESI-MS (MeOH, 250oC): (+) m/z =632 [ M + Na ]; (Appendix 20) 4.5 Synthesis of 6-maleimideocaproic acid (9) O O O O O + H2N O O OH OH 65% N O OH 9 Procedure 1 6-aminohexanoic acid (0.669 g, 5.1 mmol, 1equiv) and maleic anhydride (0.5 g, 5.1 mmol, 1equiv) in acetic acid (20 ml) were stirred at r.t. for 15 h under argon. The resulting white suspension was then heated to 150℃ for 8 h (white suspension was dissolved). The solvent was removed in vacuo, and the residue was purified by silica gel column chromatography (CHCl3: MeOH = 100:1.5). The crude product then was 38 Chapter 4 Experimental dissolved in DCM and extract by DI water three times to remove residual acetic acid. 591 mg of white solid 12 was isolated corresponding to 59% yield. Procedure 2 6-Aminohexanoic acid (1.34 g, 10.2 mmol) was added to a stirred solution of maleic anhydride (1 g, 10.2 mmol) in acetic acid. A white solid precipitated immediately and stirring was continued for 3 h at room temperature. The solid was collected by filtration. Without further purification, the product was mixed with 9 ml of acetic anhydride (9 ml). Sodium acetate (0.441 g, 5.38 mmol) was added and the reaction mixture was heated to 90℃ for 2 h. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (20 ml). The solution was washed with water (20 ml) followed by brine (20 ml) and the organic layer was dried over sodium sulfate. The solvent was removed by evaporation to provide an off-white solid, which was purified by short column chromatography (EtOAc: Hexane = 1:1) to give 6maleimideocaproic acid as a white solid. The yield was 65 %. 1 H NMR (500MHz, CDCl3): δ6.69(s, 2H), 3.52(t, 2H, 6.95Hz), 2.35(t, 2H, 7.25Hz), 1.67(q, 2H, 7.77Hz), 1.61(q, 2H, 7.57Hz), 1.34(m,2H); (Appendix 21) 39 Chapter 4 13 Experimental C NMR (500MHz, CDCl3): δ178.60, 170.84, 134.07, 37.59, 33.59, 28.16, 26.12, 24.10; (Appendix 22) 4.6 Synthesis of Tert-butyl 2, 2’-(4-(2-tert-butoxyallyl)-10-(6-(2, 5-dioxo-2, 5-dihydro1H-pyrrol-1-yl) hexanoyl)-1, 4, 7, 10-tetraazacyclododecane-1, 7-diyl) diacetate (10) Procedure To 6-maleimideocaproic acid 9 (25 mg, 0.1166 mmol, 1.2 equiv) dissolved in 1ml of DMF HBTU (59 mg, 0.1554 mmol, 1.6 equiv) and DIEA (32 µl, 0.1943 mmol, 2 equiv) were added at 0℃. The reaction mixture was stirred for 10 mins before DO3tBu 3 (50 mg, 0.09714 mmol, 1 equiv) was added and the resulting mixture was stirred overnight at r.t. The solvent was reduced under high vacuum, and the product was dissolved in DCM. The organic phase was washed with NaOH, brine and dried over 40 Chapter 4 Experimental anhydrous Na2SO4. The solvent was evaporated under vacuum and the product was purified by column chromatography (CHCl3: MeOH = 10:1) yielding 27 mg (40 %) of 10. 1 H NMR (500MHz, CDCl3): δ6.67(s, 2H), 3.64(t,2H), 3.51(m, 4H), 3.30(m, 6H), 2.99(t, 2H), 2.89(s, 2H), 2.82(s, 2H), 2.75(s, 2H), 2.28(t, 2H), 1.62(m, 4H), 1.44(t, 27H), 1.32(m, 2H); (Appendix 23) ESI-MS (MeOH, 250oC): (+) m/z= 708 [M+H]; (Appendix 24) 4.7 Synthesis of Folate-NHS (11) Procedure: Folic acid (100 mg, 0.226 mmol) was dissolved in 6 ml of dry DMF to which DCC (31 mg, 0.152 mmol) and NHS (26 mg, 0.226 mmol) were added. The reaction mixture was stirred for 20 h at r.t. in the dark. The byproduct, dicyclohexylurea, was filtered off, and 30% acetone in diethyl ether (20 ml) was added under stirring. A yellow precipitate was formed and collected on a sintered funnel; after washing with acetone and 41 Chapter 4 Experimental ether several times, the folate-NHS 11 (109 mg) was isolated and characterized by LCIT-TOF-MS (Appendix 26). 4.8 Synthesis of Folate-DOTA (13) Procedure The folate-NHS (11, 50 mg, 0.09294 mmol) was dissolved completely in 20 ml of dry pyridine. 10 ml were taken from it to a RBF and then, DO3tBu (20 mg, 0.03886 42 Chapter 4 Experimental mmol) was added. The mixture was stirred at r.t. in the dark for 48 h. After pyridine was evaporated, the resulting compound was dissolved in 3 ml TFA to remove BOC. Deprotection was carried out at r.t. for 4 h and thereafter, TFA was removed under vaccum[66]. The resulting compound was dissolved in DI water and 5 drops of TFA (5 ml). The precipitate was filtered through a #4 Pyrex sintered glass funnel, the solution was collected and water was removed under high vacuum. Since folate-DOTA (13) is water-soluble in its protonated form, cation exchange chromatography was used for purification. Procedure of cation exchange chromatography Dowex resin (2 g) was added to a large glass chromatography column and the resin was conditioned by washing three times with 1 N NH4OH (15 ml), once with 500 ml of water, once with 1 N HCl (15 ml) and once more with 500ml of water. The column eluent should be kept slightly acidic (pH 6–7). The crude folate-DOTA was dissolved in 5 ml DI water with 2 drops of TFA and added to the column; the flowthrough was collected and passed over the column once more. Wash the column with 800 ml of water or until the pH of the eluent remains constant (pH 6–7). The Folate-DO3tBu was eluted 43 Chapter 4 Experimental from the column by adding 1 N NH4OH ( 50 ml). Aqueous NH4OH was removed using a rotary evaporator at 50℃ and 8mg of a light-brown solid was isolated and characterized by LC-IT-TOF-MS [67]. LC-IT-TOF-MS (Appendix 27): (+) m/z= 770 [M+H] 4.9 Synthesis of Folate-DO3tBu (12) Procedure Folic acid (43 mg, 0.09714 mmol, 1 equiv) dissolved in 5 ml of DMF was combined with HBTU (59 mg, 0.1554 mmol, 1.6 equiv) and DIEA (32 µl, 0.1943 mmol, 2 equiv) at 0 ℃. The reaction mixture was stirred for 10 min before DO3tBu (50 mg, 44 Chapter 4 Experimental 0.09714 mmol, 1 equiv) was added. The resulting mixture was stirred again overnight at r.t.. Thereafter, the solvent was removed under high vacuum and the residue was washed with 10 ml acetone (30%) in ether, DCM (30%) in ether and water three times separately. The mixture was monitored by LC-IT-TOF-MS (Appendix 29). Time Percentage of acetonitrile 0 to 10 min 0% 13 to 24 min 20 % 30 to 33 min 30 % 45 to 47 min 60 % 52 to 60 min 100 % 60 to 63 min 0% Table 1 Eluting gradient used in Prep-HPLC. According to the LC profile, folate-DO3tBu (12) was successfully separated by GILSON Prep-HPLC (Waters XTerra Prep column, 5 µm 19*50 mm, 10 ml/min flow rate) by using water and acetonitrile as eluting solvents. The eluting gradient is shown in the table 1 and the product was characterized by LC-IT-TOF-MS (Appendix 28). 45 Chapter 4 Experimental 4.10 Synthesis of 1-[3-(4-carboxy-phenyl)-3-oxo-propyl]-piperidinium HCl (14) [61] Procedure 4-acetylbenzoic acid (500 mg, 3.045 mmol, 1 equiv), piperidine HCl (370 mg, 3.045 mmol, 1 equiv) and paraformaldehyde (274 mg, 9.135 mmol, 3 equiv) were added to 2 ml absolute ethanol. To this solution, concentrated HCl was added (30 µl). Then the mixture was heated to 105℃ for reflux. After 4 h, paraformaldehyde (274 mg, 9.135 mmol, 3 equiv) was added and the reaction was refluxed for further 6 h. Acetone was introduced into the reaction mixture to precipitate the product. Then the white precipitate was filter off and dried under the vacuum to afford the product (14) in 48% yield. 1 H NMR (300MHz, DMSO-d6): δ 1.5–1.8 (6H), 3.23(s, 4H), 3.38 (t, 2H,), 3.69 (t, 2H), 8.10 (m, 4H) (Appendix 1) ESI-MS (MeOH, 250oC): (-) m/z=260 [M-HCl] (Appendix 2) 46 Chapter 4 4.11 Experimental Synthesis of Bis-disulfide (15) [61] Procedure To a solution of ethanol (1.2 ml), methanol (0.8 ml), mannich salt (350 mg, 1.17 mmol, 1 equiv) and 4-methylbenethiol (291mg, 2.348 mmol, 2 equiv) were added. Then piperidine (0.05 ml) and 37% (wt/vol) aq. formaldehyde 0.35 ml were introduced sequentially. The reaction mixture was heated to 105 ℃ for reflux. After 1 h, aq. formaldehyde (0.35 ml, 37 %, (wt/vol)) was added via a pipette through the top of the condenser. The reaction mixture was allowed to reflux for a further 3 h. The mixture was cool to RT and and then the solvent was evaporated at 40 °C. The resulting mixture was stored in a refrigerator (4 °C) overnight. The solid was formed and washed by methanol two times. The white solid was dried under vacuum to afford product with 45% yield. 1 H NMR (500MHz, CDCl3): δ 2.38 (s, 6H), 3.16–3.31 (m, 4H), 3.85 (q, 1H), 7.15 (d, 4H), 7.18 (d, 4H), 7.64(d, 2H), 8.07 (d, 2H); (Appendix 3) 47 Chapter 4 13 Experimental C NMR (500MHz, CDCl3): δ 200.50, 137.24, 131.55, 131.15, 130.17, 129.86, 128.29, 45.88, 36.41, and 21.10; (Appendix 4) ESI-MS (MeOH, 250oC): (-) m/z=435 [M-H]; (Appendix 5) 4.12 Synthesis of 4-(3-tosyl-2-(tosylmethyl)propanoyl)benzoic acid (16) Procedure 1 A 1:1 methanol-deionized water solution (5 ml) was prepared in a 10 ml round bottom flask. To this solution, bis-sulfide (50 mg, 0.1145 mmol), and OXONE (0.422 g, 0.687 mmol) were added The reaction mixture was stirred at RT for 24 h and monitored by TLC plate using EA/Hexane (v/v=1:1) mixed solvent. The mixture was poured into a separatory funnel and extracted with chloroform twice. Then, sufficient DI-water was added in order to dissolve the inorganic salts and extract twice. The organic extract was 48 Chapter 4 Experimental combined and extracted with Brine once. The solvent was removed under vacuum to afford the product in 60 % yield. 1 H NMR (500MHz, CDCl3): δ 2.49 (s, 6H), 3.48–3.66 (m, 4H), 4.40 (q, 1H), 7.37 (d, 4H), 7.70–7.73 (m, 6H), 8.10 (d, 2H); (Appendix 6) ESI-MS (MeOH, 250oC): m/z = 499[M-H]-, 523[M+Na]+; (Appendix 7) Procedure 2 H5IO6 (22 mg, 0.0962 mmol, 2.1 equiv) was dissolved in acetonitrile (0.5 ml) by vigorous stirring at r.t., for 1 h. Then CrO3 (0.46 mg, 0.004581 mmol, 0.1 equiv) was added to the solution. The mixture was stirred at r.t. for 5min to give a clear orange solution. The H5IO6/CrO3 solution was then added dropwise over a period of 15 min to a solution of bis-sulfide (20 mg, 0.04581 mmol, 1 equiv) in ethyl acetate (1 ml) at -35℃. After the addition was completed, the reaction mixture was stirred at -35℃ for 1 h. The reaction was quenched by addition of saturated Na2SO3 solution. 49 Chapter 4 Experimental The reaction mixture contained the intermediate (17), the bis-sulfone (16) and starting material bis-sulfide (15). It’s more complicated than OXONE oxidation, so that OXONE was chose as oxidation agent at last. 4.13 Synthesis of N-(2-bromoethyl)-4-(3-(p-tolylthio)-2-((p-tolylthio) methyl)propanoyl)benzamide (18) Procedure 2-bromoethylamine hydrobromide (28 mg, 0.1374 mmol, 1.2 equiv) and DIEA (23 µl, 0.1374 mmol, 1.2 equiv) were added into 0.5ml DCM. Then, the mixture is stirred until the 2-bromoethylamine hydrobromide is completely converted into 2bromoethanamine, as monitored by TLC using DCM/MeOH (v/v=10:1) mixed solvent. 50 Chapter 4 Experimental In the meanwhile, bis-sulfide (15, 50 mg, 0.1145 mmol, 1 equiv), EDC (33 mg, 0.1718 mmol, 1.5 equiv) and DMAP (2 mg, 0.01718 mmol, 0.15 equiv) are under vacuum for 30 min. Then, argon was introduced first and followed by adding 0.5 ml DCM. After the bissulfide has reacted to form the intermediate (monitored via TLC), a freshly prepared solution of 2-bromoethanamine was added. The reaction mixture is stirred at RT overnight, then poured into a separatory funnel and extracted with NaHCO3 twice and Brine once. The organic extract was combined and the solvent was removed. The mixture was purified by chromatography column using EA/Hexane (v/v=2:1) as eluting solvent combination. After drying under vacuum, the 39mg product was identifided as a mixture of N-(2-bromoethyl)-4-(3-(p-tolylthio)-2-((p-tolylthio)methyl)propanoyl)benzamide (18) and 2-(4-(3-(p-tolylthio)-2-((p-tolylthio)methyl)propanoyl)benzamido)ethan-1-ylium (22). 1 H NMR (500MHz, CDCl3): δ 7.88(d, 2H, 8.45Hz), 7.56(d, 2H, 8.45Hz), 7.13(d, 4H, 7.75Hz), 7.05(d, 4H, 8.05Hz), 4.47(t, 2H, 9.63Hz), 4.10(t, 2H, 9.5Hz), 3.78(m, 1H, 7.5Hz), 3.24(q, 2H, 7.03Hz), 3.15(q, 2H, 6.57Hz), 2.34(s, 6H). (Appendix 8) ESI-MS (MeOH, 250oC): (+) m/z = 462 [M - Br]+ ; (Appendix 9) Fab-MS : (+) m/z = 462 [M - Br]+, 565[M+Na]+; (Appendix 10) 51 Chapter 4 4.14 Experimental Synthesis of N-(2-(piperazin-1-yl)ethyl)-4-(3-(p-tolylthio)-2-((p- tolylthio)methyl)propanoyl)benzamide (23) Procedure Bis-sulfide 15 (20 mg, 0.04581 mmol, 1 equiv) dissolved in 1ml of DMF, HBTU (28 mg, 0.0733 mmol, 1.6 equiv) and DIEA (15 µl, 0.09162 mmol, 2 equiv) were added together at 0 ℃ . The reaction mixture was stirred for 10 min before 2piperazinoethylamine (18 mg, 0.1374 mmol, 3 equiv) was introduced and the resulting mixture was stirred overnight at r.t.. The solvent was reduced under high vacuum, and the product was dissolved in DCM. The organic phase was washed with NaHCO3, brine and dried over anhydrous Na2SO4 and evaporated under vacuum. Since the desired piperazine bis-sulfide 23 was form as the minor product, this method was also aborted. 52 Chapter 4 4.15 Experimental Synthesis of Tert-butyl 4-(2-aminoethyl) piperazine-1-carboxylate (26) Procedure [68] In a flask equipped with a Dean-Stark trap and a condenser, a mixture of 2piperazinoethylamine (3.6 ml, 27.45 mmol) in 4-Methyl-2-pentanone(MIBK, 55 ml, 2 L/M) was heated to reflux under argon. Reaction progress was monitored by recording the volume of water (0.5 ml) produced. After no more water was produced, the mixture was cooled to 0℃. Boc anhydride (6 g, 27.45 mmol) dissolved in a minimum of MIBK was then added dropwise to the flask. After stirring for 0.5 h at r.t., water (5.5 ml, 0.2 L/M) was added. The aqueous layer was separated, and MIBK was evaporated under reduced pressure leading to the imine intermediate 25. Water (3 ml) and 2-propanol (28 53 Chapter 4 Experimental ml) were then added, and the mixture was heated to 50℃ until completion of the hydrolysis. Solvents were then distilled off providing the clean side product 27. 1 H NMR (500MHz, CDCl3): δ 1.46 (s, 18H, 1.90Hz), 2.39 (t, 4H, 5.02Hz), 2.46 (t, 2H, 6.00Hz), 3.23(m, 2H, 5.05Hz), 3.42 (t, 4H, 5.2Hz); (Appendix 12) ESI-MS (MeOH, 250oC): (+) m/z= 330 (M+H); (Appendix 13) 4.16 Synthesis of Tert-butyl 4-(2-aminoethyl) piperazine-1-carboxylate (31) 4-(3-(p-tolylthio)-2-((p-tolylthio)methyl)propanoyl)benzoic acid (15, 30mg, 0.06871mmol, 1equiv) dissolved in 1ml of DMF was added HBTU (42mg, 0.1099mmol, 1.6equiv) and DIEA (23μl, 0.1374mmol, 2equiv) at 0℃. The reaction mixture was stirred for 10mins before DO3tBu (3, 39mg, 0.07558mmol, 1.1equiv) was added. The resulting mixture was stirred overnight at r.t.. The solvent was reduced under high vacuum, and the product was dissolved in Chloroform. The organic phase was washed with DI water, 54 Chapter 4 Experimental brine and dried over anhydrous Na2SO4. The crude product was purified by chromatography column using Chloroform/Methanol (v/v=80:1) as eluting solvent combination. The solvent was removed under vacuum to afford the product in 12 % yield. 1 H NMR (500MHz, CDCl3): δ 7.57(d, 2H), 7.32(d, 2H), 7.16(d, 4H), 7.06(d, 4H), 3.78(m, 3H), 3.58(t, 2H), 3.31(d, 4H), 3.25(q, 2H), 3.14(q, 2H), 3.09(s, 2H), 3.02(t, 2H), 2.89(t, 2H), 2.77(m, 6H), 2.67(s, 2H), 2.34(s, 6H), 1.44(t, 27H). (Appendix 25) 55 Chapter 5 Chapter 5. Conclusion Conclusion and future work The focus of this thesis was to synthesis suitable imaging agents with specific selectivity via two major strategies: (1) The synthesis of intercalators that allow the functionalization of proteins or peptides by reacting with disulfide bridges and (2) the synthesis of the chelator DOTA bearing a single functionality as well as the combination with the cell targeting entity folic acid that facilitates cell-specific cell uptake. The bis-disulfone (16) had been synthesized successfully. This molecule allows the specific functionalization of proteins or peptides by intercalating into disulfide bonds. A major drawback of such intercalators is their low solubility in aqueous solution and the necessity to use organic solvents such as DMSO or acetonirile, which strongly limits its application for the functionalization of proteins with relatively low stability. Therefore, novel, less lipophilic intercalator molecules had been designed and the reaction schemes for their preparation had been presented in this thesis. Despite various attempts, the targeted molecule was not synthesized and isolated. In another section, the mono-functionalization of the compexing agent DOTA achieved was reported. Furthermore, a single ethynyl as well as maleimido group were also successfully incorporated into DOTA. This important building block allowed the 56 Chapter 5 Conclusion straigntforward preparation of the targeted Folate-DOTA conjugate This “bifunctional molecule” offers folic acid as the cell targeting entity and the chelator DOTA that can interact with a range of metal and transition metals e.g. gadolinium or gallium, which had been which could be used for PET or MRT imaging. Such a molecule is highly attractive for imaging tissue of cancer cells that overexpress folic acid receptors such as certain breast cancers. Future experiments will be conducted with the company Siemens in order to further assess the suitability of this molecule for PET imaging of cancer tissue. It would be very appealing to conjugate the intercalator synthesised with a DOTA or folic acid group in order to decorate therapeutically relevant proteins or peptides with the additional functionalities. However, in this case water-solubility of the intercalator remains a key problem. Therefore, more water soluble intercalators need to be designed and synthesized The synthesis of the somatostatin DOTA conjugate was not achieved due to the degradation of the DOTA-intercalator, 31, under oxidizing condition. However, we proposed here an alternative synthetic strategy where the bis-disulfone (16) can be directly coupled with DO3tBu (3) (Scheme 15). The biological properties of the somatostatin-DOTA conjugate can then be investigated by in vitro experiments. 57 Chapter 5 Conclusion Scheme 15. Proposed synthetic route for somatostatin DOTA conjugate Based on this approach, a similar strategy can be used to label larger proteins bearing several disulfide bonds with the prospect of attaching a larger number of gadolinium complexes, which may ultimately improve the contrast and detection limit to a greater extent. 58 Chapter 6 References Chapter 6. References 1. Willmann, J.K., et al., Molecular imaging in drug development. Nature Reviews Drug Discovery, 2008. 7(7): p. 591-607. 2. Massoud, T.F. and S.S. Gambhir, Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes & Development, 2003. 17(5): p. 545-580. 3. Bartholoma, M., et al., Single amino acid chelates (SAAC): a strategy for the design of technetium and rhenium radiopharmaceuticals. Chem Commun (Camb), 2009(5): p. 493-512. 4. Bowen, M.L. and C. Orvig, 99m-Technetium carbohydrate conjugates as potential agents in molecular imaging. 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Bianchi, A., et al., Thermodynamic and structural properties of Gd3+ complexes with functionalized macrocyclic ligands based upon 1,4,7,10- tetraazacyclododecane. Journal of the Chemical Society-Dalton Transactions, 2000(5): p. 697-705. 10. Li, C., et al., Multimodal Image-Guided Enzyme/Prodrug Cancer Therapy. Journal of the American Chemical Society, 2006. 128(47): p. 15072-15073. 11. Nicolle, G.M., et al., From monomers to micelles: investigation of the parameters influencing proton relaxivity. Journal of Biological Inorganic Chemistry, 2002. 7(7-8): p. 757-769. 12. Jaszberenyi, Z., et al., Physicochemical and MRI characterization of Gd3+loaded polyamidoamine and hyperbranched dendrimers. Journal of Biological Inorganic Chemistry, 2007. 12(3): p. 406-420. 13. Kamaly, N., et al., Bimodal Paramagnetic and Fluorescent Liposomes for Cellular and Tumor Magnetic Resonance Imaging. Bioconjugate Chemistry, 2007. 19(1): p. 118-129. 14. Na, H.B., I.C. Song, and T. 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European Journal of Nuclear Medicine and Molecular Imaging, 2006. 33(9): p. 1007-1016. 32. Mathias, C.J., et al., Synthesis of 99(m)Tc DTPA-folate and its evaluation as a folate-receptor-targeted radiopharmaceutical. Bioconjugate Chemistry, 2000. 11(2): p. 253-257. 33. Siegel, B.A., et al., Evaluation of In-111-DTPA-folate as a receptor-targeted diagnostic agent for ovarian cancer: Initial clinical results. Journal of Nuclear Medicine, 2003. 44(5): p. 700-707. 62 Chapter 6 34. References Mathias, C.J., et al., Receptor-mediated targeting of Ga-67-deferoxamine-folate to folate-receptor-positive human KB tumor xenografts. Nuclear Medicine and Biology, 1999. 26(1): p. 23-25. 35. Mathias, C.J., et al., Preparation of Ga-66- and Ga-68-labeled Ga(III)deferoxamine-folate as potential folate-receptor-targeted PET radiopharmaceuticals. Nuclear Medicine and Biology, 2003. 30(7): p. 725-731. 36. Bettio, A., et al., Synthesis and preclinical evaluation of a folic acid derivative labeled with F-18 for PET imaging of folate receptor-positive tumors. Journal of Nuclear Medicine, 2006. 47(7): p. 1153-1160. 37. Sun, C., R. Sze, and M.Q. Zhang, Folic acid-PEG conjugated superparamagnetic nanoparticles for targeted cellular uptake and detection by MRI. Journal of Biomedical Materials Research Part A, 2006. 78A(3): p. 550-557. 38. Choi, H., et al., Iron oxide nanoparticles as magnetic resonance contrast agent for tumor imaging via folate receptor-targeted delivery. Academic Radiology, 2004. 11(9): p. 996-1004. 39. Mathias, C.J., et al., Preparation of 66Ga- and 68Ga-labeled Ga(III)deferoxamine-folate as potential folate-receptor-targeted PET radiopharmaceuticals. Nuclear Medicine and Biology, 2003. 30(7): p. 725-731. 40. Patel, Y.C., Somatostatin and its receptor family. Front Neuroendocrinol, 1999. 20(3): p. 157-98. 41. Froidevaux, S. and A.N. Eberle, Somatostatin analogs and radiopeptides in cancer therapy. Biopolymers, 2002. 66(3): p. 161-83. 63 Chapter 6 42. References Evers, B.M., et al., Somatostatin and analogues in the treatment of cancer. A review. Ann Surg, 1991. 213(3): p. 190-8. 43. Kratz, F., Albumin as a drug carrier: Design of prodrugs, drug conjugates and nanoparticles. Journal of Controlled Release, 2008. 132(3): p. 171-183. 44. Kochendoerfer, G.G., Site-specific polymer modification of therapeutic proteins. Current Opinion in Chemical Biology, 2005. 9(6): p. 555-560. 45. Antos, J.M. and M.B. Francis, Transition metal catalyzed methods for siteselective protein modification. Current Opinion in Chemical Biology, 2006. 10(3): p. 253-262. 46. McFarland, J.M., N.S. Joshi, and M.B. Francis, Characterization of a threecomponent coupling reaction on proteins by isotopic labeling and nuclear magnetic resonance spectroscopy. J Am Chem Soc, 2008. 130(24): p. 7639-44. 47. Gauthier, M.A. and H.A. Klok, Peptide/protein-polymer conjugates: synthetic strategies and design concepts. Chem Commun (Camb), 2008(23): p. 2591-611. 48. Davis, B.G., Chemical modification of biocatalysts. Current Opinion in Biotechnology, 2003. 14: p. 379-386. 49. Romanini, D.W. and M.B. Francis, Attachment of Peptide Building Blocks to Proteins Through Tyrosine Bioconjugation. Bioconjugate Chemistry, 2007. 19(1): p. 153-157. 50. Antos, J.M. and M.B. Francis, Selective Tryptophan Modification with Rhodium Carbenoids in Aqueous Solution. Journal of the American Chemical Society, 2004. 126(33): p. 10256-10257. 64 Chapter 6 51. References Joshi, N.S., L.R. Whitaker, and M.B. Francis, A Three-Component Mannich-Type Reaction for Selective Tyrosine Bioconjugation. Journal of the American Chemical Society, 2004. 126(49): p. 15942-15943. 52. Kodama, K., et al., Site-Specific Functionalization of Proteins by Organopalladium Reactions13. ChemBioChem, 2007. 8(2): p. 232-238. 53. Carrico, I.S., Chemoselective modification of proteins: hitting the target. Chem Soc Rev, 2008. 37(7): p. 1423-31. 54. Pavlou, A.K. and J.M. Reichert, Recombinant protein therapeutics[mdash]success rates, market trends and values to 2010. Nat Biotech, 2004. 22(12): p. 1513-1519. 55. Leung, H.J., et al., Impact of an easily reducible disulfide bond on the oxidative folding rate of multi-disulfide-containing proteins. J Pept Res, 2005. 65(1): p. 4754. 56. Petersen, M.T.N. and P.H. Jonson, Amino acid neighbours and detailed conformational analysis of cysteines in proteins. Protein Engineering, 1999. 12: p. 535-548. 57. Betz, S.F., Disulfide bonds and the stability of globular proteins. Protein Sci, 1993. 2(10): p. 1551-8. 58. Zloh, M., et al., Identification and insertion of 3-carbon bridges in protein disulfide bonds: a computational approach. Nat Protoc, 2007. 2(5): p. 1070-83. 59. Thornton, J.M., Disulphide bridges in globular proteins. Journal of Molecular Biology, 1981. 151(2): p. 261-287. 65 Chapter 6 60. References Brocchini, S., et al., Disulfide bridge based PEGylation of proteins. Advanced Drug Delivery Reviews, 2008. 60(1): p. 3-12. 61. Brocchini, S., et al., PEGylation of native disulfide bonds in proteins. Nat Protoc, 2006. 1(5): p. 2241-52. 62. Li, C. and W.-T. Wong, A Simple, Regioselective Synthesis of 1,4-Bis(tertbutoxycarbonylmethyl)- tetraazacyclododecane. The Journal of Organic Chemistry, 2003. 68(7): p. 2956-2959. 63. Prasuhn De Jr Fau - Yeh, R.M., et al., Viral MRI contrast agents: coordination of Gd by native virions and attachment of Gd complexes by azide-alkyne cycloaddition. (1359-7345 (Print)). 64. Li, C. and W. T. Wong, A selective one-step synthesis of tris N-alkylated cyclens. Tetrahedron, 2004. 60(26): p. 5595-5601. 65. Machitani, K., et al., Molecular design of tetraazamacrocyclic derivatives bearing a spirobenzopyran and three carboxymethyl moieties and their metal-ion complexing behavior. Analytical Sciences, 2008. 24(4): p. 463-469. 66. Dhar, S., et al., Targeted Single-Wall Carbon Nanotube-Mediated Pt(IV) Prodrug Delivery Using Folate as a Homing Device. Journal of the American Chemical Society, 2008. 130(34): p. 11467-11476. 67. Link, A.J., M.K.S. Vink, and D.A. Tirrell, Synthesis of the functionalizable methionine surrogate azidohomoalanine using Boc-homoserine as precursor. Nat. Protocols, 2007. 2(8): p. 1884-1887 66 Chapter 6 68. References Laduron, F., et al., Efficient and Scalable Method for the Selective Alkylation and Acylation of Secondary Amines in the Presence of Primary Amines. Organic Process Research & Development, 2004. 9(1): p. 102-104. 67 Appendix Appendix: NMR and MS Data 1.1HNMR of 1-[3-(4-carboxy-phenyl)-3-oxo-propyl]-piperidinium HCl (14) 68 Appendix 2. ESI-Mass spectrum (-) of 1-[3-(4-carboxy-phenyl)-3-oxo-propyl]-piperidinium HCl (14) 69 Appendiix 3. 1 HMR of 4-(3-(p-tolyltthio)-2-((p-ttolylthio)meethyl)propannoyl)benzoiic acid (15) S O S HO O 70 Appendix 4. 13 CNMR of 4-(3-(p-tolylthio)-2-((p-tolylthio)methyl)propanoyl)benzoic acid (15) 71 Appendix 5. ESI-mass spectrum of 4-(3-(p-tolylthio)-2-((p-tolylthio)methyl)propanoyl)benzoic acid (15) 72 Appendix 6. 1 HNMR of 4-(3-tosyl-2-(tosylmethyl)propanoyl)benzoic acid (16) 73 Appendix 7. ESI-MS data of 4-(3-tosyl-2-(tosylmethyl)propanoyl)benzoic acid (16) 74 Appendix 8. 1 HNMR of N-(2-bromoethyl)-4-(3-(p-tolylthio)-2-((p-tolylthio)methyl)propanoyl)benzamide 75 Appendix 9. ESI-MS data of N-(2-bromoethyl)-4-(3-(p-tolylthio)-2-((p tolylthio)methyl)propanoyl) benzamide 76 Appendix 10. Fab-MS data of N-(2-bromoethyl)-4-(3-(p-tolylthio)-2-((p tolylthio)methyl)propanoyl) 77 Appendix 78 Appendiix 11. LC-IT-TOF F-MS spectruum of piperrazine-bis-sulfide file: LC profi MS specctrum of Peak 1 Inten.(x10,000 0) 498.57 737 7.5 5.0 2.5 246.14 432 136.0429 9 424.1959 0.0 250 0 500 0 750 1000 1250 1500 1750 m /z 1000 1250 1500 1750 m /z MS specctrum of Peak 2 00) Inten.(x100,00 2.5 465.8776 6 2.0 1.5 1.0 697.8150 0.5 0.0 250 0 500 0 750 MS specctrum of Peak 3 79 Appendix 12. 1H NMR of tert-butyl 4-(2-(tert-butoxycarbonylamino)ethyl)piperazine-1-carboxylate 80 Appendix 13. ESI-MS spectrum of tert-butyl 4-(2-(tert-butoxycarbonylamino)ethyl)piperazine-1carboxylate 81 Appendix 14. 1HNMR of DO3tBu (3) 82 Appendix 15. 13CNMR of DO3tBu (3) 83 Appendix 16. ESI-MS of DO3tBu (3) 84 Appendix 17. 1HNMR of 2-bromo-N-(prop-2-ynyl) acetamide (6) 85 Appendix 18. ESI-MS of 2-bromo-N-(prop-2-ynyl) acetamide (6) 86 Appendix 19. 13C NMR of tert-butyl 2,2',2''-(10-(2-oxo-2-(prop-2-ynylamino)ethyl)-1,4,7,10tetraazacyclododecane-1,4,7-triyl)triacetate (8) 87 Appendix 20. ESI-MS of tert-butyl 2,2',2''-(10-(2-oxo-2-(prop-2-ynylamino)ethyl)-1,4,7,10tetraazacyclododecane-1,4,7-triyl)triacetate (8) 88 Appendix 21. 1HNMR of 6-maleimideocaproic acid (9) 89 Appendix 22. 13CNMR of 6-maleimideocaproic acid (9) 90 Appendix 23. 1HNMR of maleimido-DO3tBu (10) 91 Appendix 24. ESI-MS of maleimido-DO3tBu (10) 92 Appendix 25. 1HNMR of tri-tert-butyl 2,2',2''-(10-(4-(3-(p-tolylthio)-2-((p-tolylthio)methyl)propanoyl) benzoyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (31) 93 Appendiix 26. LC-IT-TOF L F-MS spectru um of folatee-NHS file LC profi The IT-T TOF-MS sp pectrum of Peak P 4 The IT-T TOF-MS sp pectrum of Peak P 5 94 Appendiix 27. LC-IT-TOF L F-MS spectru um of folatee-DOTA (fr from the firsst proceduree) file LC profi m AU 400 00 214nm ,4nm (1 .00) 300 00 200 00 100 00 0 0.0 2 2.5 5.0 0 7.5 10.0 12.5 15.0 17.5 m in The corrresponding IT-TOF-MS spectrum L F-MS spectru um of folatee-DOTA (fr from the seccond proceddure) 28. LC-IT-TOF LC profi file m AU(x100) 254nm ,4nm (1.0 0) 2.5 5 2.0 0 1.5 5 1.0 0 0.5 5 0.0 0 -0.5 5 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 m in 95 Appendix The corresponding IT-TOF-MS spectrum 29. LC-IT-TOF-MS spectrum (x10,000,000) STIC 2.25 1:TIC 2:TIC 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 2.5 5.0 7.5 10.0 12.5 15.0 17.5 LC profile 96 Appendix Inten.(x1,000,000) 401.295 2.5 289.174 0.0 250 500 750 1000 1250 1500 1750 m /z 1750 m /z 1750 m /z The MS spectrum of the first peak in LC profile Inten.(x1,000,000) 515.357 5.0 2.5 347.175 0.0 250 500 750 1000 1250 1500 The MS spectrum of the second peak in LC profile Inten.(x100,000) 1.0 629.436 0.5 461.266 288.183 0.0 250 500 750 1000 1250 1500 The MS spectrum of the third peak in LC profile 30. The LC-IT-TOF-MS spectrum m AU(x1,000) 254nm ,4nm (1.00) 1.5 1.0 0.5 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 m in LC profile 97 Appendix Inten.(x1,000,000) 4.0 469.7405 3.0 2.0 295.0809 441.7103 1.0 385.6478 0.0 200 300 400 938.4721 500 600 700 800 900 1000 The MS spectrum of the last peak in the LC profile 98 [...]... can be engineered into the protein as point mutations using molecular biological techniques Such approaches are usually demanding and expensive and point mutation may have a negative impact on protein function by altering its structure Moreover, introducing an accessible free thiol group often leads to disulfide scrambling, protein misfolding and an increased tendency to form aggregates during purification... receptors 8 Chapter 1 1.4 Introduction Chemical Modification of Proteins There is an increasing interest in protein conjugates for diagnosis and therapy Even though proteins often display limited pharmacokinetics, low proteolytic stabilities and the possibility to elicit immune responses, there have been successful attempts of converting proteins into efficient drug delivery systems or imaging agent [43] Their... modify proteins involves the introduction of non-canonical amino acids (rNCAA) into proteins[45] Here, by chemically attaching the desired rNCAA to suppressor tRNA and then placing the amber codon at the desired position in the mRNA, a number of rNCAA have been incorporated at different positions into the protein sequence Successful examples of rNCAA that are incorporated into the protein sequence include... prevent disulfide scrambling reactions if more than one disulfide bond is reduced[61] 1.6 Design of biocompatible metal- complex protein conjugates In order to design biocompatible metal- complex protein conjugates which can achieveprecise intracellular delivery and improved sensitivity for imaging, two components are required, namely, an imaging agent and an efficient targeting entity There are several... highly defined structures, biodegradability and the presence of a high number of functional groups available for chemical modifications make them attractive for applications in targeted drug delivery and bioimaging Figure 5 (a) Site-specific modification of protein yields homogenity and (b) nonspecifcification modification of protein results heterogeneity[44] 9 Chapter 1 Introduction Protein conjugates... been made in recent years in the development of targeted contrast agents for diagnostic imaging that allows better differentiation [14, 15] Various targeted contrast agents for MRI and PET have been reported which were synthesized via the conjugation of metal chelates to various biomolecules, including peptides[16], proteins[17], antibodies[18], oligonucleotides[19] and biotin/avidin[20] These biomolecules... Introduction Metal Complex for MRI or PET Molecular imaging is one of the most exciting and rapidly growing areas of science as it enables the characterization and quantification of biological processes at the cellular and subcellular level in living subjects in an intact manner[1] It utilizes specific molecular probes as well as intrinsic tissue characteristics as the source of image contrast, and offers the... imaging probes which show high binding affinity to the target receptors, antigens, and nucleic acids being specifically overexpressed in or on the targeted cells tissues 3 Chapter 1 1.2 Introduction The Biological Function of Folic Acid Folic acid(Figure 1) is a water-soluble vitamin of the B -complex group and plays essential roles in numerous bodily functions by participating in the biosynthesis of. .. opportunity for an improved understanding of integrative biology, earlier detection and characterization of diseases, and facilitates a better evaluation of therapeutic treatment[2] The imaging modalities can be broadly divided into two categories: anatomical and molecular techniques Examples of anatomical imaging technologies include computed tomography (CT) and magnetic resonance imaging (MRI), which... Targeting particular cells or tissues for imaging e.g proliferative cells or for transporting drug molecules plays a vital role in cancer treatment and represents an area of high scientific interest In order to contribute to a better detection of proliferative cells, a sophisticated metal- DOTA imaging agent was designed that is able to specifically interact with disulfide bridges of proteins by intercalating .. .Design and Synthesis of novel metal complex- protein conjugation agents for Applications in bio- imaging WANG TAO (BSc, Sichuan University2008 ) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF. .. Modification of Proteins There is an increasing interest in protein conjugates for diagnosis and therapy Even though proteins often display limited pharmacokinetics, low proteolytic stabilities and the... Design of biocompatible metal- complex protein conjugates In order to design biocompatible metal- complex protein conjugates which can achieveprecise intracellular delivery and improved sensitivity for